JP2002315589A - Gene of helicobacter pylori needed for urease control and maturation and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene that is needed for controlling and maturing urease in Helicobacter pylori and the like. SOLUTION: The objective gene is an isolated nucleic acid corresponding to the nucleic acid sequence that is one part of the nucleic acid sequences represented by Figures 4A through 4I and corresponds to the genes called ureE, ureF, ureG, ureH and ureI or to modified sequences thereof, or an isolated nucleic acid that is at least an arbitrary part of these nucleic acid sequences excluding the part of the nucleic acid sequence of the ureI gene corresponding to the nucleotide 209-282 represented in the Figures 4A through 4I in an arbitrary fragment in the part of the ureI gene (wherein as for the specific properties of polypeptides UreE, UreF, UreG, UreH or UreI wherein H. pylori will be expressed, the functional property of the polypeptide encoded by the above- stated nucleic acid parts is retained, reduced or deleted).

Description

DETAILED DESCRIPTION OF THE INVENTION

Helicobacter pylori (also referred to as H. pylori ) is a gram-negative bacterium that is presently found exclusively in the gastric mucosa of humans, more particularly only on the surface around the crater foci of gastric and duodenal ulcers It is. This bacterium was originally called Campylobacter pyloridis [Warren et al. (1983) Lancet 1, 1273-127.
Five〕.

[0002] Like most bacteria, H. pylori is acidic.
Sensitive to medium at pH, but still able to tolerate acidity in the presence of physiological proportions of urea [Marsha
ll et al. (1990) Gastroenterol. 99 : 697-702]. By hydrolyzing urea in the form of carbon dioxide and ammonia, which is salted out in the bacterial microenvironment, it is assumed that the urease of H. pylori will allow the bacteria to survive in the acidic environment of the stomach. Have been. More recently, studies performed on animal models have provided elements that suggest that urease is a key factor in gastric mucosal colonization (Eaton et al. (1991) Infect. . 5
9 : 2470-2475]. Urease is also thought to cause direct or indirect damage to the gastric mucosa.

Helicobacter pylori ( H. pylori ) is currently recognized as a causative agent of gastric inflammation of the antrum and appears to be one of the cofactors required for ulcer development. Also, it appears that gastric carcinoma development can be linked to the presence of H. pylori .

[0004] All strains clinically isolated from biopsies or gastric juice are one of the most immunogenic proteins of H. pylori.
It synthesizes highly active urease (urease) exposed on the surface of bacteria. Urease may play a role in the etiological process, which means that strains with poor urease-producing ability obtained by chemical mutagenesis can colonize the stomach of pigs. This has been recognized by experiments performed on pigs, indicating that none were present. Nevertheless, these results obtained after chemical mutagenesis failed to colonize the stomach because other genes could be inactivated during generalized mutagenesis. Does not make it possible to say for certain that it is due to a reduction in urease production. Therefore, this is not a controllable mutation, so this technique has practical advantages in devising measures to reduce and thus prevent the deleterious effects of urease in the case of infection by H. pylori. Not shown.

In addition to its role in gastric colonization,
It has been shown that urease and free ammonia can have a direct cytotoxic effect on epithelial cells and an indirect effect by inducing an inflammatory response responsible for gastric lesions.

[0006] Urease is therefore one of the most important virulence determinants, of H. pylori specifically inactivated in genes associated with urease expression, whether structural or accessory. Construction of isogenic strains to limit the role of urease in the colonization stage and to apply it to, for example, the construction of strains that can be used to protect individuals in the vaccination process by constructing attenuated strains, It is of great importance.

[0007] Until now, the urease gene was H. pylor.
localized on a 34 kb fragment of the i chromosome,
It has also been linked to the 4.2 kb region present in this fragment. This 4.2 kb region contains ure
Four genes, referred to by the terms A , ureB , ureC and ureD , have been linked. This area is called Camp
When a 4.2 kb DNA was transferred via a shuttle vector in ylobacter jejuni , it was possible to obtain a urease-positive phenotype.

However, the transformation of E. coli cells with the 4.2 kb DNA described above did not make it possible to obtain expression of urease activity in E. coli .

[0009] The present inventor determines what factors are necessary for expressing urease activity in E. coli as obtained in H. pylori from the viewpoint of culture conditions as well as from the viewpoint of genetics. Succeeded. The present inventors have, in this respect, the expression of urease in E. coli is, E.
It was determined that the activation of the nitrogen regulatory system in E. coli was dependent on the presence of an accessory gene for the structural gene for urease. The inventor has also referred to the expression "auxiliary gene" of urease in the following, E. col.
allowing functional expression of urease in a i, and H. p
Several genes that determine urease maturation and regulation in ylori have been identified and isolated.

Accordingly, the present invention relates to H. pylori and E. col
a collection of five new genes that may play a decisive role or at least intervene in the functional expression of urease in i , as well as each of these genes considered separately and independently of the other genes . The invention is likewise described in the publications (Labigne et al. (1.
991) J. Bacteriol. 173 : 1920-1931], urease
It also relates to an optionally altered collection of these genes in association with the structural genes designated ureA , ureB , ureC and ureD .

[0011] The present invention also relates to compositions which can be utilized for protection against infection by the new in vitro detection means and H. pylori infection by H. pylori.

Accordingly, the present invention corresponds to the following nucleotide chains: ureE , ureF , ureG , ure
H , at least one corresponding to a gene called ureI
It is intended a nucleotide sequence constituted or characterized by one nuclear sequence or at least one part of at least one of these nuclear sequences.

[0013]

[Table 1]

[0014]

[Table 2]

[0015]

[Table 3]

[0016]

[Table 4]

[0017]

[Table 5]

[0018]

[Table 6]

[0019]

[Table 7]

[0020]

[Table 8]

[0021]

[Table 9]

The nucleotide sequence according to the present invention is DN
A or RNA.

[0023] The present invention also provides that the functional properties of the polypeptide encoded by the altered gene are similar to those of H. pylor.
polypeptide UreE, Ur as expressed by i
one or more nucleotides in such a way as to be retained or attenuated or deleted in relation to the properties of eF, UreG, UreH or UreI, or such that the sequence does not express the polypeptide in H. pylori . Deletion,
It also relates to nucleotide sequences that have been altered in the context of the above nucleotide sequences by additions, substitutions or inversions.

According to a particular embodiment of the invention, within the above-defined frame, the nucleotide sequence is: a) corresponding to the nucleotide chain shown in FIGS. 4A to 4I, ureE , ureF , ureG , ureH , Ur
a collection of nuclear sequences corresponding to genes called eI , or b) a collection formed by nuclear sequences (mutants) corresponding to these genes which have been independently modified, and Is a polypeptide UreE, UreF, UreG, UreH as expressed by H. pylori
Or a polypeptide having functional homology to UreI, or conversely, a polypeptide UreE, UreF, UreG, Ur as expressed by H. pylori
It is characterized by being constituted by or including a modified polypeptide-encoding form which attenuates and / or eliminates the functional properties of eH or UreI.

The fragments (nucleotide chains) of the above-mentioned nucleotide sequences are advantageous for a variety of reasons, for example the following may be mentioned:

UreE , ure in H. pylori
A fragment of the above sequence, which retains the ability to encode a polypeptide having functional homology to the polypeptide as obtained by expression of a gene selected among F , ureG or ureI ;

Encoding any part of the above-mentioned polypeptide as obtained in H. pylori , in particular H.
a fragment encoding a part of a peptide or polypeptide that can be recognized by an antibody directed against pylori or behave as a hapten or immunogen;

The genes ureE , ureF , ure
G, as expressed from ureH or ureI such H. pyl
a fragment of the above sequence which is not capable of encoding an ori polypeptide;

H. pylori genes ureE , ure
F , a fragment that encodes a polypeptide or peptide having attenuated or even deleted properties in relation to the properties of the polypeptide encoded by ureG , ureH or ureI .

Such a fragment advantageously has at least 15, preferably at least 20, nucleotides.

These genes ureE , ureF , ur
eG , ureH and ureI are on the chromosome of H. pylori ; these genes are so-called auxiliary genes in relation to the structural genes for urease ( ureA , ureB ). Contrary to the structural genes, auxiliary genes are not required for urease enzyme formation. Conversely, these genes do not intervene in the functional expression of urease as expressed in H. pylori by means of regulation and / or maturation of the formed urease. Urease is actually expressed in the form of an inactive apoenzyme before undergoing a maturation step within H. pylori, a step that confer a functional enzyme form.

The present inventor has also found that the presence of these five auxiliary genes indicates that the structural genes ureA , ureB , ure
C and ureD were identified as essential for the expression of functional urease in E. coli cells previously transformed.

Thus, the identification of these genes and their nucleotide sequences makes it possible to examine the means for modulating the urease activity of the strain of H. pylori , in particular for preparing attenuated strains.

According to a first embodiment of the invention, the advantageous nucleotide sequence is the natural polypeptide UreE, U
It encodes a polypeptide having functional homology to reF, UreG, UreH and UreI. These homologies between the polypeptides correspond to the native polypeptide UreE,
As UreF, UreG, UreH and UreI, H.
It is evaluated in relation to the ability of these polypeptides to function inside pylori and thus contribute to the formation of functional urease from apoenzymes.

[0035] This functional homology can be detected by using the following tests: i.e., 10 ⁹ bacteria urea 1 ml - resuspended in indole medium and incubated at 37 ° C.. The hydrolysis of urea results in the release of ammonia, thus increasing its pH, thereby triggering a color change from orange to fushia red.

On the contrary, within the framework of the present invention, the polypeptide which it encodes no longer has the ability of the native polypeptide to enable the production of functional urease in H. pylori or, in some cases, in another species. The genes ureE , ureF , ureG , ur
A nucleotide sequence corresponding to a collection of nuclear sequences corresponding to eH or ureI can be used. In this case, one seeks to attenuate or eliminate the functional properties of the native polypeptide as expressed by H. pylori . Functional properties are considered to have been attenuated if the strain into which the nucleotide sequence according to the invention has been inserted produces a non-pathogenic urease, for example in the form of an apoenzyme. This virulence can be assessed using the following tests:

Eaton et al. (1991, Infect. Immun. 59 : 2470-)
2475), the animal, preferably gnotobiotic (gnotobiotiqu
e) Test the transfer of the recombinant strain into the piglet stomach.

According to a first embodiment of the present invention, the nucleotide sequence as described above comprises the structural genes ureA and ur encoding the urease subunit in H. pylori .
It can be associated with the nuclear sequence corresponding to eB .

[0039] According to another embodiment of the present invention, the nucleotide sequence, the gene ureA encoding urease in H. pylori, ureB, ureC and / or <br/> is associated with UreD.

In this case, the various genes can be located on completely different replicons.

The present invention likewise falls within the scope of the above definition and comprises the genes ureE , ureF , ureG , u
It also relates to the nucleotide sequence corresponding to one of the coding nucleotide chains corresponding to reH or ureI . In this regard, the invention particularly relates to the following chains:

A strand ureE corresponding to nucleotides 800 to 1309 of the sequence of FIGS. 4A to 4I or under stringent conditions, ie 6 × SSC Denhardt (Denh
ard) any fragment of this strand when hybridized with the strand ureE or a sequence complementary thereto at 68 ° C. or in 5 × SSC-50% formamide at 37 ° C. in a medium;

A chain ureF corresponding to nucleotides 1324 to 2091 of the sequence of FIGS. 4A to 4I, or 37 under stringent conditions, ie, in 6 × SSC Denhard's medium at 68 ° C. or 5 × SSC-50% formamide. In ° C., any fragment of this chain when hybridized with linked ureF or a sequence complementary to this chain,

A chain ureG corresponding to nucleotides 2123 to 2719 of the sequence of FIG. 4A to FIG. 4I or 37 under stringent conditions, ie, in 6 × SSC Denhard's medium, at 68 ° C. or in 5 × SSC-50% formamide ° C, any fragment of this strand when hybridized with the strand ureG or a sequence complementary to this strand,

A chain ureH corresponding to nucleotides 2722 to 3516 of the sequence of FIGS. 4A to 4I, or 37 g under stringent conditions, ie, in 6 × SS Denhard's medium at 68 ° C. or 5 × SSC-50% formamide. ° C, any fragment of this strand when hybridized with the strand ureH or a sequence complementary to this strand,

A strand ureI corresponding to nucleotides 211 to 795 of the sequence of FIGS. 4A to 4I, or 6 g under stringent conditions, ie in 6 × SS Denhard medium.
At 8 ° C. or in 5 × SSC-50% formamide
Any fragment of this strand when hybridized at 7 ° C. with strand ureI or a sequence complementary to this strand.

Here, "complementary sequence" in relation to the DNA sequence
What is referred to as inverted and complementary sequences.
The term "reverse" refers to the 5'-3 'of a nucleic acid that is nucleotide in nature complementary in relation to a given sequence.
Considering the recovery of orientation.

The present invention also provides a GCG AAA ATA TGC TAT GAA AT
It also relates to a specific nucleotide chain corresponding to the sequence A GGA AAC CGC CAT.

The invention likewise relates to any DNA sequence comprising this nucleotide chain.

A nucleotide sequence according to the invention corresponding to the above definition, if labeled at the 5 'and / or 3' end with a substance to be detected, can be included in the construction of a probe. . Labels include radioisotopes, enzymes, chemical or chemiluminescent labels, fluorescent dyes,
Haptens or antibodies, base analogs and even physical labels can be mentioned. These labels can optionally be immobilized on a solid support, such as a granular or membrane-like support such as a magnetic sphere.

An example of a preferred label is radioactive phosphorus ( ³² P) incorporated at the 5 ′ end of the sequence used as a probe.

Advantageously, a nucleotide probe according to the invention comprises any fragment of the above-mentioned gene, for example a fragment of about 45 nucleotides.

A preferred probe according to the present invention, genetic <br/> child ureH or preferably is composed of fragments derived from genes UREI.

[0054] from the nucleotide sequence in accordance with the present invention, H.
Primers that can be used for in vitro detection of infection by pylori can also be constructed. The primer is characterized in that it contains a nucleotide fragment as derived from the aforementioned sequence, comprising about 18 to about 30, preferably about 25 to about 30 nucleotides. Such primers can be used, for example, in a gene amplification reaction according to a chain polymerization technique.

For use in amplification techniques, the primers of the invention are taken in pairs under defined conditions so as to hybridize to the 5 'and 3' ends of the nucleotide fragment to be amplified, respectively. .

When utilizing the PCR technique, the conditions required for specific hybridization of the primer with the DNA to be detected are described in EP-A-200363,
No. 201184, No. 229701, and the temperature is calculated according to the following formula: T (° C.) = [4 (C + G) +2 (A + T) −10] where A, T, C, G represent the number of nucleotides A, T, C, G in the primers used, respectively.

Amplification techniques that can be used within the framework of the present invention include:
For example, the European patent application of Cetus (2003003, 2
P.01184 and 229701)
CR (Polymerase Chain Reaction) technology or Biotechnolog
y (Vol. 6, October 1988), and the like.

Other nucleotide sequences according to the invention are those which hybridize under the stringent conditions described above to the sequences defined above or to sequences complementary to these sequences.

[0059] Nucleotide sequences and vectors of the present invention, Likewise, other genes or sequences of H. pylori, or other strains, H. pylori, or E. coli, in other hosts such as adenovirus It can also be used for expression.

The present invention further relates to the polypeptides UreE, UreF, UreG, U shown in FIGS.
It also relates to a polypeptide characterized in that it corresponds to one of reH or UreI, or any part of at least one of these polypeptides. The invention particularly relates to UreE, UreF, UreG, UreH or Ur as expressed by H. pylori.
is altered immediately when it exhibits functional homology to a polypeptide from eI, or conversely, is altered by deletion, addition, substitution or inversion of one or more amino acids,
It relates to any polypeptide that attenuates or even deletes its functional properties, such as urease activity as expressed by H. pylori .

The polypeptides UreE, UreF, Ure
G, UreH and UreI intervene, among other things, in the regulation and maturation of urease in H. pylori .

Another polypeptide according to the invention is Ala
Lys Ile Cys Tyr Glu Ile Gly Asn Arg His 11
It corresponds to a chain of amino acids.

The polypeptides of the invention, especially those polypeptides whose sequences are given above, may be used for the production of polyclonal or monoclonal antibodies or for the detection of antibodies in biological samples infected with H. pylori . Available for

Monoclonal antibodies can be prepared by known techniques for preparing human antibodies or by hybridoma technology. These antibodies are also available from Marks
It can also be prepared using the technique described by J. Mol. Biol. 1991 222 , 581-597.

The present invention also relates to anti-idiotype antibodies. Antibodies against the 11 amino acid chain described above can be utilized in the context of the urease maturation blocking reaction.

The present invention further relates to the use of monoclonal or polyclonal antibodies in a composition for treating infection by H. pylori .

The present invention also aims at a recombinant vector characterized by containing the DNA sequence of the present invention. Such a recombinant vector may be, for example, a cosmid or a plasmid.

A vector which is particularly advantageous for the practice of the present invention is that it is known on October 3, 1991, under the number I-1148, CNCM [Collection Nationale de Cultures de Microorga, Paris, France.
nisms)] and the plasmid pILL753 contained in E. coli HB101.

Another particularly advantageous recombinant vector is the one which has been identified on October 3, 1991, under the number I-1149, under the name C-1149.
It is characterized by being plasmid pILL763 contained in E. coli HB101 deposited with NCM.

The present invention is also directed to a recombinant host cell (or recombinant cell line) characterized by being transformed with a nucleotide sequence satisfying the above definition. The host cell transformed in this way must allow expression of the nucleotide sequence of the urease auxiliary gene, optionally modified according to the above definition.

In a preferred example, the recombinant host cell is characterized in that the product of the altered accessory gene altered by one of the nucleotide sequences described above, or advantageously self-expressed, has the effect of urease, especially H. p modified in a manner that contributes to attenuating its pathogenic effect
ylori strain.

For example, such a recombinant strain is known as 19
On June 26, 1992, the NCIMB (National Collections of Indus
H. pylor deposited with the strial and Marine Bacteria LTD)
i , which can be obtained by mutation of the N6 strain, wherein the mutation is the gene ureE , ureF , ur
It is performed at the level of at least one of eG , ureH or ureI and / or at the level of one or more structural genes, for example ureA or ureB .

Preferably, within the framework of the present invention, according to the criteria defined previously, a recombinant strain with reduced urease activity, in particular a H. pylori strain, is formed.

Thus, a particularly advantageous recombinant N6 strain is
The urease-negative phenotype can be obtained and the mutated genes ureE , ureF , ureG ,
ureH or ureI .

Inactivation of the gene ureI makes it possible, for example, to prepare urease-negative H. pylori strains. Similarly, while the products of the genes ureA and ureB are expressed, some mutations within ureI allow the urease negative phenotype to be obtained in H. pylori . For example, the mutation No. described in the examples. 8 is that.

In particular, vaccine strains, especially vaccines H. p
Another mutation that is particularly advantageous for the preparation of ylori strains is
It is a mutation of the gene ureG . A recombinant H. pylori strain in which the gene ureG has been mutated exhibits the following properties: -The mutated strain retains the ability to mount an immune response. The strain carrying such a mutation does not have urease activity.

However, it is possible to transform other strains with the sequences according to the invention. In particular, the gene u previously inserted into this strain via, for example, a plasmid
To achieve mutations in reE , ureF , ureG , ureH or ureI , E. coli will be utilized. The gene thus mutated can then be introduced into another host cell, for example, H. pylori , to allow allelic replacement and create a mutation.

Deletion of the gene ureI in a recombinant E. coli cell according to the present invention does not alter this phenotype when other conditions for the expression of the urease-positive phenotype are met. Please note that.

The recombinant E. coli strain may further comprise the polypeptide UreE, UreF, UreG, UreH or UeE.
It can be used to produce reI and to purify them by conventional techniques.

Recombinant strains of H. pylori with reduced urease activity can likewise be used for the transport and expression of heterologous genes, such as, for example, the cholera and Salmonella genes.

Various techniques can be used to realize a recombinant strain. For example, electroporation as described in the examples of the present application is utilized.

In some cases, the electroporation method can be modified by eliminating the step consisting of applying an electric shock at the level of the cells to be transformed.

The present invention is particularly directed to a means for protecting against infection by H. pylori by administration of an immunogenic composition comprising a recombinant cell line characterized by an attenuated urease activity. I will provide a. Such immunogenic compositions are available in human medicine.

The immunogenic composition may optionally comprise genes ureE , u which have been modified to reduce urease activity.
a strain, such as a cell of H. pylori , wherein urease activity has been attenuated by the insertion of a nucleotide sequence according to the present invention into the strain, comprising at least one sequence corresponding to reF , ureG , ureH or ureI. Good.

In general, what may be problematic here is, for example, one or more genes ureA , ureB , ure
C , ureD , ureE , ureF , ureG , ure
Any host capable of producing an attenuated urease by mutation of the nucleotide sequence of H or ureI , or by expression of a partially truncated form of the polypeptide that intervenes in the structure, maturation or regulation of urease.

The present invention also provides a kit for the in vitro diagnosis of infection by H. pylori on a defined biological sample: ureE , ureF , ureG , ureH or ur
at least one corresponding to a gene selected from eI
5 'of a nucleotide fragment specific for one nuclear sequence
And at least one pair of nucleotide primers that can hybridize to the 3 'end, meeting the above criteria,-the reagents necessary to extract nucleic acids from the processed sample,-the amplification of the fragment that one wishes to amplify. A sufficient amount of reagents for carrying out the polymerization of said nucleotide fragments from nucleotide primers, in particular polymerases, which can be used as probes, and which hybridise under defined conditions with the amplified DNA fragments Also intended is a kit, characterized in that it comprises at least one nucleotide chain, possibly a means for revealing hybridization.

According to one particular embodiment of the invention, the kit may also include, for example:-by including a resistance gene for antibiotics or by being composed of chromosomal DNA of N6; Consisting of nucleic acids, possibly carried by one plasmid, which can be easily detected by hybridization, said fragment further comprising at least two amplification primers at these two ends, these primers being the primers according to the invention. An amplification reaction internal control, selected or not selected from among the following:-a probe capable of hybridizing with the nucleic acid contained in the internal control;-optionally, in the sample to be tested. Reverse transcriptase for obtaining cDNA from RNA that is optionally present; It can also be done.

[0088] Due to the presence of the internal control added to the sample,
It is possible to detect the presence of "false negatives" in the sample. In fact, if the internal control specific probe does not detect amplification products, it is likely that H. pylori DNA or cD
There are samples that contain inhibitors of Taq polymerase, which are inhibitors of NA amplification. In this case, various dilutions of the sample to be tested make it possible to demonstrate the presence of H. pylori nucleic acids.

If the internal control shows a positive response, it can be concluded by the negative reaction at the level of the sample tested that H. pylori is just absent.

It should be noted that the primer incorporated into the internal control is not necessarily the primer of the present invention. However, if other primers are selected, the sensitivity may decrease.

As an example of a biological sample for detecting human infection by H. pylori, a collected sample such as a biopsy, gastric juice, or, in some cases, saliva or stool is used. The kit can also be used for water contamination testing or food testing.

The present invention also provides for H. p
A method for the in vitro diagnosis of infection by ylori, a) under conditions to provide accessibility in the form of single-stranded DNA or RNA, a nucleic acid sample that may contain H. pylori, H contacting with at least one pair of nucleotide primers according to the present invention capable of hybridizing to the pylori nucleic acid, if present, and initiating the synthesis of a primer extension product, wherein H pylori nucleotide sequence serving as a template when combined with a primer; b) separating the synthesized nucleic acid strand from the template; c) detecting a sufficient amount of the desired nucleic acid to be detected. Until amplification is obtained, the nucleic acids which can hybridize with the primers and are present at the end of step b) The step of repeating the synthesis of extension products from the strands, under conditions to be able to detect the presence of amplified nucleic acids d) are sought, the nucleotide probe and step c)
And e) detecting any hybridization product that has been formed.

According to a preferred embodiment of the method for in vitro diagnostics described above, prior to contacting the sample to be tested, there is a step of processing the sample, such as extracting nucleic acids.

According to another preferred embodiment, the method comprises the step of including any RNs present in the sample to be tested.
One step is included prior to the step of contacting with the primer, consisting of treating the nucleic acid of the sample with reverse transcriptase to synthesize cDNA from A.

[0095] The present invention is also, in the infection by H. pylori
In a kit for in vitro diagnostics: -an amount of a probe as defined above; -a medium suitable for performing a hybridization reaction between the nucleic acid of H. pylori to be detected and the probe; -optionally formed A kit comprising a reagent for detecting a hybrid.

The use of this kit, and a method for the in vitro diagnosis of infection with H. pylori based on biological samples, comprises the steps of: preparing a sample to be tested whose DNA and / or RNA has been made accessible beforehand; Contacting the probe with the probe under conditions that permit hybridization of the nucleic acid with the probe;-characterizing an optional hybridization reaction between the nucleic acid and the probe.

The nucleotide sequence of the present invention is H. pylori
Of the nucleic acid and digestion and purification with a selected endonuclease, or by chemical synthesis.

As an example, for the synthesis of such nucleic acid fragments, Narang, SA et al.
ymol., 68 , 90 (1979). Another method adapted for the preparation of nucleotide fragments is Brow
n The phosphotriester method as described by EL et al. in Meth. of Enzymol., 68 , 109 (1979).

The preparation can likewise be carried out by automated methods, for example by intervening diethylphosphoramidite as a starting component, in which case the synthesis is carried out according to Beaucage et al., Tetrahedron Letters (1981),
22 , 1859-1862.

Materials and Methods Identification of I-Genes Bacterial Strains, Plasmids and Culture Conditions H. pylori 85P was isolated from patients suffering from gastritis.
This is described by Labigne et al. (J. Bacteriol. 173 : 1920-1931 (199
1)]. E. coli MC1061 was used as a host in the cloning experiment.
[Maniatis et al. (1983), Molecular Cloning: A Laboratory Manua
l) Cold Spring Harbor Laboratory, Cold Spring Ha
utilizing Rbor NY], as the host for the quantitative analysis of urease expression, E. coli HB101 (HsdR h
sdM reA supE44 lacZ4 LeuB
6 proA2 thi-1

Sm ) [Boyer et al. (1969) J. Mol. Bio
l. 41 : 459-472]. The vectors and hybrids used in this study are listed in Table 1. E. coli
The strain was transformed into L-broth without glucose (per liter,
10 g of tryptone, 5 g of yeast extract and 5 g of NaCl,
(pH = 7.0) or in a box of L gelose (containing 1.5% gelose) at 37 ° C. Antibiotic concentrations for selection of transformants were as follows (in milligrams per liter): kanamycin: 20, tetracycline: 8, ampicillin: 1
00, spectinomycin: 100, carbenicillin:
100. For the expression of urease activity, 0.4% D-glucose as a carbon source and 0.2% freshly prepared by filtration sterilization unless otherwise indicated, as a nitrogen source.
(Weight / volume) of L-glutamine [Pahel et al. (1982)
J. Bacteriol. 150 : 202-213), the E. coli bacteria were cultivated on a medium with a limited nitrogen source concentration, consisting of M9 minimal medium with gelose without ammonium (pH = 7.4).

Molecular Cloning and DNA Analysis Digestion with restriction endonucleases, end filling and other general DNA manipulations were performed according to standard techniques of Maniatis et al. [Maniatis et al. (1983), Molecular Cloning, Experimental Room Manual, Cold Spring Harbor Lab
oratory, ColdSpring Harbor NY]. Partial digestion with Sau3A was performed at 20 ° C. in a manner to suppress enzyme activity. Restriction endonucleases, large (large) fragment of DNA polymerase I, T4 DNA polymerase (used to blunt the ends of the fragments) and T4 DNA ligase were supplied by Amersham Corp. Calf intestinal alkaline phosphatase, Phar
Sourced by macia. DNA fragments were separated by electrophoresis on a horizontal block of gel containing 1% or 1.4% agarose and treated in Tris-acetate or Tris-phosphate buffer [Maniatis et al. (1983) ),
Molecular Cloning, Laboratory Manual, ColdSpring Har
bor Laboratory, Cold Spring Harbor NY]. A 1 kb scale was used as a standard for molecular weight (Bethesda Resea
rch Laboratories). Ethidium bromide (0.4μg / ml)
The electroelution of DNA fragments from agarose gels containing was performed as described previously [J. Bacteriol. 173:
1920-1931 (1991), Labigne et al.].

Urease activity Urease activity was detected using a urea-indole medium (Diag
nostic Pasteur) resuspension of 10 ⁹ bacteria in a 1 ml,
And incubation at 37 ° C. for varying times. Release of ammonia by urease activity increased the pH and induced a color change from orange to red.

According to the above-mentioned change of working mode, [Ferrero
et al. (1991) Microb.Ecol.Hlth.Dis. 4: 121-134),
Urease activity was measured by the Berthelot reaction. Briefly, bacteria were collected from the gelose box in 2.0 ml of sterile 0.85% NaCl and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. 0.
The precipitate was washed twice in 85% NaCl, 10 mM EDTA
100 mM sodium phosphate buffer (pH 7.4) containing
(PEB). To prepare sonicated extracts, cells were lysed using a BransonSonifier Model 450 adjusted to 30 w, 50% cycle with four 30 second impulses. Cell debris was removed before measuring urease. Freshly prepared samples (10-5
0 μl) was added to 200 μl of a urea substrate solution (50 mM urea prepared in PEB) and allowed to react for 30 minutes at normal temperature. The reaction was stopped by adding 400 μl of the phenol-nitroprusside reagent and 400 μl of the alkaline hypochlorite reagent. The reaction mixture was incubated at 50 ° C. Blanks in which urease activity was inactivated by boiling for 5 minutes prior to substrate addition were treated in a similar manner. The amount of free ammonia was determined from a standard curve for setting the relationship between the A ₆₂₅ and the concentration of ammonium (from NH ₄ Cl). The release of 2 μmol of ammonia is 1 μm
It was considered equivalent to the hydrolysis of mol urea. Urease activity was expressed in μmoles of urea hydrolyzed per minute per mg of bacterial protein.

Protein Determination According to the Bradford test (Sigma Chord)
emicals), the protein concentration was measured. To solubilize the protein in the whole cell extract, the cell suspension prepared in TPE was centrifuged, and the sediment was washed with octyl-β-D-
The final surfactant concentration (in dye reagent) was 0.1-0.2% (weight / volume) by resuspension in the glucopyranosides solution.

Transposon Mutagenesis and Mutations
Construction of variants A mini Tn3-Km feeding system was used to generate mutations by random insertion into a DNA fragment cloned into pILL570. Plasmid pOX38 as a transposable element donor and plasmid pTCA acting on trans to supply the enzyme transposase Tn3 [Seifert et al. 1985, Genetic Engineer
ing Principles and Methods, Volume 8: p123-134, Setlow, J. and Hollaeinder,
A. Editors, PlenumPress New York]
(1986, PNAS USA 83: 735-739) and the Cre Tn encoding the mini Tn3 system, as well as the recombinase (recombinase) P1 specific for the lox site.
Strain NS2114 harboring the gene was utilized for mutagenesis of DNA fragments with the following changes:

I) Plasmid pTn (Seifert et al.
al., 1986), the BglI-EcoRI fragment of the gene encoding β-lactamase was removed and this was replaced with the kanamycin cassette ClaI-C. jejuni [Labigne-
Roussel et al. (1988, J. Bacteriol. 170: 1704-1708)
Mini Tn3 was changed. This new insertion factor Mini Tn3-Km was translocated into the transferable plasmid pOX38 as described by Seifert et al. (1986, supra), which induced the acquisition of plasmid pILL553.

Ii) For cloning of the fragments used for mutagenesis, Labigne et al. (1991, J. Ba.
cteriol. 173: 1920-1931), using the conjugated spectinomycin pILL570 suicide vector described previously. This suicide vector was constructed from pILL560 (Labi) lacking DNA sequences associated with immunity to Tn3.
gne-Roussel et al., 1988, J. Bacteriol. 170: 1704-
1708).

Iii) Plasmid IncP, pRK212.1 of "complementary plasmid" (Figurski et al., 1979,
PNAS USA 76: 1648-1652) E. coli NS2 by conjugation
NS221 harboring the cre gene introduced into 114 strains
Four spontaneous rifampicin mutants were obtained and used for the selection of transzygotes harboring co-integrates.

Iv) 500 μg of kanamycin and 300
The 3rd obtained on a medium containing μg of spectinomycin
PILL by depositing a mixture of
Due to the large number of copies of the plasmid derived from 570, effective segregation of co-integrants (product of co-integration) was selected by positive selection.

DNA Sequencing To read the two complementary strands independently, M1
3mp19 and M13mp18 [Meissing et al. (1982)
Gene 19 : 269-276]. Clones containing the insert were identified using X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and isopropyl-β-D-thiogalactopyranoside.
Recombined plasmids M13mp18 and M13mp
19 single-stranded by the polyethylene glycol method [Sanger e
tal. (1980) J. Mol. Biol. 143 : 161-178]. Required Sequenase (United States Biochemical cor
p.) and the chain terminator method using dideoxynucleotides [Sanger et al. (1977) Proc. Nat.
Sequence was performed according to l. Acad. Sci. USA 74 : 5463-5467]. The double-stranded DNA was similarly sequenced by the chain terminator method using dideoxynucleotides using the required sequence, utilizing plasmid DNA purified on a cesium chloride gradient [Zhang et al. (1988) ) Nucleic Acids Research 16 : 12
20]. A sample of 3 μg of DNA was first denatured with a 1 M NaOH solution (20 μl total volume) and then neutralized with 2 μl of 2 M ammonium acetate (pH 4.6). After adding 60 μl of 100% frozen ethanol, incubating at −70 ° C. for 10 minutes and centrifuging at 4 ° C. for 20 minutes,
The DNA was precipitated. 80% frozen ethanol 60
After washing with μl, the sediment was resuspended in 10 μl of sequencing buffer containing 0.5 pmo of primer and
Incubated for minutes. After a 30 minute incubation time at normal temperature, sequencing was performed.

Results The E. coli strain harboring the recombinant cosmid pILL585
Detection of urease activity in host strain E. coli transformants harboring cosmid pILL585 were spread on M9 minimal or L medium with glucose supplemented with 0.2% 1-glutamine (as sole nitrogen source). , 37 ° C for 48 hours. next,
Transformants were screened for urease activity according to a qualitative colorimetric test performed in urea-indole medium. Activity was observed only in E. coli HB101 transformants that had undergone multiple passages (5 passages or more) on minimal medium at 37 ° C. under aerobic conditions. Therefore, this is the condition used for qualitative measurement of urease expression in E. coli clones. No urease activity was detected in transformants cultured on nitrogen rich media.

A 4.2 kb fragment identified as the minimum region required for expression of urease in C. jejuni in E. coli cells, even after culture and passage in media limiting the concentration of the nitrogen source. (Labigne et al. (1991) J. Bac
teriol. 173 : (1920-1931)]
Transformation of E. coli HB101 strain at 590. This implies that the gene present on the cosmid but lacking in the plasmid pILL590 is required for urease expression in E. coli .

[0114] Remains Required for Urease Activity of E. coli Strains
E. coli harboring the recombinant subcloned plasmid pILL590
In the absence of detectable urease activity in the strain, the 34 kb insert of cosmid pILL585 was partially digested with the endonuclease Sau 3A to produce a 7-12 kb fragment. These fragments were treated with alkaline phosphatase to avoid entirely the relocation of the beginning of the genome (rearrangement), and ligated into plasmid pILL570 was linearized with Bam HI. After transformation in E. coli HB101, each transformant resistant to spectinomycin was subjected to subsequent tests for its ability to hydrolyze urease under inducing conditions. 1
One clone exhibited a urease-positive phenotype. It contained a recombinant plasmid called pILL753. This plasmid contained an insert fragment of 11.2 kb. In relation to the sole restriction sites Eco RI and Pst I vector pILL570, Ba
The mHI and HindIII recognition sites were mapped (FIG. 1). By comparing the restriction map of plasmid pILL753 with the restriction map of the recombinant plasmid described above, p
The insert fragment of ILL753 is transformed into plasmid pIL
The four genes for urease previously identified in L590 (ie, ureA , ureB , ureC and u
reD ) was found to have an additional 4.6 kb DNA fragment downstream.

Urease activity in E. coli HB101
To define the culture conditions to ensure optimal expression of the genes of the urease H. pylori in sexual optimization E. coli, after culture on minimal medium supplemented with various nitrogen sources, pILL753
The activity of clones harboring was evaluated quantitatively. In each case, solid minimal basal medium was used, as studies have shown that urease activity is very low in cultures performed in liquid medium.

The relative activities of cultures in medium supplemented with L-arginine, L-glutamine, L-glutamate, NH ₄ Cl and urea (10 mM final concentration each) were 100%, 36%, 27%, respectively. 46% and 20%.

The urease activity was optimal in culture performed on a medium supplemented with L-arginine. Urease activity was performed on nitrogen rich media and was not detected in the culture.

[0118] The presence of free N _i ²⁺ ions is likely to have an effect of stimulating the urease activity [Mulrooney
et al. (1989) J. Gen. Microbiol. 135 : 1769-1776,
And Mobley et al. (1989) Microbiol. Rev. 53 : 85-10.
8] This did not appear for urease activity of cells harboring pILL753.

PILL cultured under various conditions
Analysis of the course of urease expression in E. coli clones carrying 753 showed that the maximum urease activity was obtained after aerobically culturing at 37 ° C. for 3 days on minimal medium supplemented with L-arginine. (Figure 2). Urease activity of cultures performed on nitrogen-rich media was highest after microaerobic culture.
Conversely, microaerobic conditions had an inhibitory effect on the activity of the nitrogen-limited culture.

PILL75 in a minimal medium supplemented with arginine at 37 ° C. for 3 days under aerobic conditions
The urease activity of E. coli cells harboring 3 was 0.9 ± 0.4 μmol urea hydrolysis per minute per mg protein. In comparison, the isolate of H. pylori used to clone the urease gene was 23.2 ± 2.
Urea was hydrolyzed at a rate of 3 μmol (μmol / min / mg protein).

Urease activity in E. coli host strains
In order to identify the genes required for cloning and to determine the DNA region required for the localization- positive urease phenotype, first the pI carrying the transposable element Mini Tn3-Km
The derivative of LL753 was isolated according to the working mode described above (see "Materials and Methods" section). All transformants of E. coli HB101 carrying the transposon were screened for urease activity. These were designated pILL753 :: x, where x points to the insertion site of Mini Tn3-Km such that it appears on the map of FIG.
Of the 24 insertions selected for analysis, 10 derivatives have completely lost the ability to hydrolyze urea (2, 3, 4, 5, 6, 10, 11, 12, 13, and 1).
4) On the other hand, 14 had a urease-positive phenotype. These results confirm that any insertion mutation (mutants 2, 3, 4, 5, and 6) having a map corresponding to the gene ureA or ureB abolishes urease activity, but similarly A 2.6 kb DNA fragment further downstream from the gene ureB is
They also show that it is required for the expression of the urease-positive phenotype in E. coli cultured under nitrogen-restricted conditions. Conversely, the results for transposon mutagenesis did not reveal that the 600 bp fragment immediately downstream of the gene ureB was essential for urease activity in E. coli .

Additional analysis, including verification of deletions in the pILL753 insert, was performed to better understand the conditions required for active urease expression in E. coli cultures. A subclone of E. coli carrying the plasmid derivative was the subject of quantitative measurement of urease activity under the above-described nitrogen-limited conditions. The results are summarized in Table 2.
The results can be compared because all subclones were derivatives of the same vector, pILL570. One of them, plasmid pILL768, was obtained by self-ligation of a large Eco RI fragment made from the restriction digest of plasmid pILL753 :: 16 (FIG. 1). This construct contains pILL7
A 2.95 kb deletion at the 3 'end of the 53 insertion segment was introduced. Cells carrying this plasmid express relatively low urease activity (Table 2). Plasmid pIL
L763 contains the ClaI- Pst of plasmid pILL753 :: 1 into the linearized vector pILL570 .
Obtained by cloning of the I restriction fragment. 1.75 kb D containing the aforementioned genes ureC and ureD
This construct, in which the NA fragment was deleted, gave pILL
753 cells expressed urease activity approximately twice as high as the urease activity. In each case, deletion or insertion did not lead to constitutive urease activity.

[0123] The necessary region for urease expression in E. coli.
Analysis of Region Sequence In a 11.2 kb fragment required for expression of urease in E. coli , a 3.2 kb DNA fragment located immediately downstream of the gene ureB was identified according to the strategy of FIG.

I) The 1.2 kb Hind III fragment and the 1.3 kb Bam HI- Hind III fragment were ligated with the DN of phages M13mp18 and M13mp19.
Plasmid pILL753 :: 12 in A, pILL7
53 :: 11, pILL753 :: 10, a) Cloning of the restriction fragment described above, b) SpH I- Ba
m HI fragment, SpH I- Hind III fragment, after c) Bam HI- Hind III fragment was sequenced independently;

Ii) Phages M13mp18 and M13m
Within the DNA of p19, the plasmids pILL753 and pILL75
Restriction fragment from ILL589 (supra), 1.2 kb Hind III, 3.8 kb Bam HI- P
stI and the 1.3 kb Bam HI- Pvu II were cloned;

Iii) Twelve oligonucleotide primers were synthesized to confirm the read and generate a sequence spanning the three independently sequenced fragments. These primers were utilized for sequencing analysis of double-stranded DNA.

Analysis of the sequence was performed using ureI , ureE , ur
Five open reading frames (ORFs), designated eF , ureG and ureH , were revealed. All of these genes are transcribed in the same direction,
It encodes peptides of 5, 170, 256, 199 and 265 amino acids. No significant length ORF was observed on the opposite complement of the sequence shown in FIGS. 4A-4I. Five ORFs are characteristic start codons A
Begins with TG. Before 4 of the 5 ORFs,
Consensus for binding to E. coli ribosomes (Sh
ine-Dalgarno) sequence [Shine et
al. (1974) Proc. Natl. Acad. Sci. USA 71 : 1342-134
6].

The upstream region of each ORF is Y = T or C, R
= G or A and Z = A or T, the sequence TGGYA
Studies have been conducted on the existence of regulatory sites for nitrogen with YRN ₄ YYGCZ [Morett et al. (1989)
J. Mol. Biol. 210 : 65-77]. A unique site was found 210 bp upstream of the locus ureG . The exact location is shown in FIGS. 4A-4I. E. col
A consensus sequence for the promoter type of i (σ70) was observed upstream of the genes ureI, ureF and ureH (TTGACA, -35, and TATAAT,
-10).

[0129]

[Table 10]

[0130]

[Table 11] (1) Bacteria cultured in an aerobic medium at 37 ° C. for 3 days on an M9 minimal medium supplemented with 0.01 M L-arginine. (2) For comparison, the urease activity of H. pylori 85P, the original isolate from which the DNA was cloned, was 23 ±
2.3 μmol urea / min / mg protein. (3) No urease activity was detected. (4) Result for specific measurement: 0.73. (5) Result for specific measurement: 0.10.

Discussion The first case of functional expression of a gene from H. pylori in a strain of E. coli is introduced here. This was possible by culturing E. coli cells harboring the pILL585 urease recombinant cosmid on a minimal medium containing a limited nitrogen source (Labigne et al.-Supra).
1991). The results obtained indicate that the urease gene of H. pylori may be under the control of the nitrogen regulatory system (NTR), and that urease activity in E. coli cells depends on the presence of the aforementioned gene assembly. Could be shown. This gene assembly is based on Labigne et
al., 1991, the four genes u
It was located just downstream of reA , ureB , ureC and ureD . These new genes are ureI , u
5 called reE , ureF , ureG and ureH
It is on a 3.2 kb fragment containing two open reading frames.

The use of mutations by insertion and deletion at the level of an 11.2 kb DNA fragment (pILL753) subcloned from the primordial cosmid allows the genes ureA , ureB , ureB , and ureB , to express urease activity in E. coli . ureF , ureG and ure
H could be required. Conversely, mutations due to insertions within the gene ureI did not significantly affect urease activity in E. coli cells. Deletion of the genes ureC and ureD (as in plasmid pILL763) results in a much stronger activity than that obtained in cells carrying the plasmid with the intact locus, It suggests a role for the cluster of H. pylori urease genes in this region.

It seems clear that pILL753 probably does not carry all of the elements necessary for full expression of urease. The main evidence is that
On the one hand, the E. coli cells harboring pILL753 had urease activity which was about 25 times weaker than that of the H. pylori isolate originally used for cloning, and the other. Thus, the deletion of the region downstream of ureH (pILL768) led to a marked decrease in urease activity. C. j
It is interesting that ejuni requires a smaller number of genes for enzyme expression than the results obtained in E. coli . Therefore, C. jejuni must be able to complement the function of the cloned gene of H. pylori .

The need for accessory genes was similarly determined by Providen
cia stuartii (Mulrooney et al. (1988) J. Bacteriol.
170 : 2202-2207), urease-positive E. coli (Collins e
t al.-1988), Klesiella pneumonia (Gerlach et al.
(1988) FEMS Microbiol. Lett. 50 : 131-135), Prote
us vulgaris (Morsdorf et al. (1990) FEMS Microbiol.
Lett. 66 : 67-74), Staphylococcus sarophyticus (Ga
termann et al. (1989) Infect. Immun. 57 : 2998-300
2), Klebsiella aerogenes (Mulrooney et al.-1990)
And Proteus mirabilis (Jones et al. (1989) J. Bact.
171 : 6414-6422 and Walz et al. (1988) J. Bacteriol.
170 : 1027-1033].

FIG. 5 shows a comparison of the three regions encoding urease for multiple bacterial species, showing the similarity and specificity of each. The degree of closeness represented by the genetic organization and the encoded polypeptide was determined by P. mir.
Between abilis and K. aerogenes , they are even more intense than their respective degrees of closeness to H. pylori . The H. pylori polypeptide UreG is derived from K. aerog
Enes polypeptide showed strong similarity to UreG (9
H. pylori and K. aerog
The degree (conserved and identical) between the enes polypeptides UreE and UreF was (33% and 14%, respectively).
%), (44% and 11.6%). Mulrooney
Have described the auxiliary proteins UreE, UreI and UreG.
It was confirmed that the K. aerogenes gene encoding is involved in activation of apoenzyme by incorporation of nickel into the urease subunit. Klebsiella and Pr
Due to the presence of a series of histidine residues at the carboxy terminus of the oteus polypeptide UreE, Mulrooney et al. proposed that UreE could interact with nickel and then transfer it to the apoenzyme. . Such a series of residues was not found in the H. pylori polypeptide UreE nor in any other product of the urease gene.

An amino acid sequence deduced from the urease gene of H. pylori and a DNA binding site (Pabo et a
l.-1981) or the ATP binding site (Higgins et al.-19).
85) Studies of similarity with the consensus sequence involved in the identification of the binding site of DNA inside the product of the gene ureI (FIGS. 4A to 4I). In addition, the well conserved ATP binding site (-GVCGSGKT
-) Is present at the NH ₂ end of the product of the gene ureG .

The urease region of H. pylori exhibits several unique elements, such as:
First, the genes ureC , ureD , and ureI are H.
peculiar to pylori . Next, the urease region is transcribed in the same direction, with 4 between ureD and ureA.
It consists of three gene blocks exhibiting an intergenic region of 20 bp and 200 bp between ureB and ureI . This suggests a unique gene organization of H. pylori , in which three gene blocks can be independently regulated.

It is generally assumed that urease synthesis by H. pylori is performed constitutively. The results presented here show that H. pylori urease gene expression
Indeed, they tend to indicate that they can be under the control of an adjustment system. In fact, once transferred into E. coli
Expression of the urease gene of H. pylori is completely under the control of the nitrogen regulatory system (NTR). The urease gene of H. pylori is the gene ntrA , n of E. coli.
Although it may be directly dependent on the synthesis of trB , ntrC products, they may depend on the expression of other gene (s) encoding one or more proteins similar to the E. coli ntr product. We cannot help but consider gender. Based on these data, it can be assumed that physiological parameters, such as the presence of a solid medium or microaerobic atmosphere, may play a role in urease expression in H. pylori in vitro or in vivo.

II. Preparation of mutant strains-strain used for electroporation experiments: strain 85P described in the aforementioned Labigne et al.-1991 publication used to carry out the initial cloning of the urease gene. Multiple strains isolated from the biopsy were tested for their electroporation suitability, including: October 3, 1991
N deposited with the CNCM on the date I-1150
Only one strain, designated No. 6, gave a positive result.

Creation of mutants in the cloned fragment of the chromosome of H. pylori strain 85P:
Mutants are prepared by mutagenesis using a transposon (Mini Tn3-Km) that allows random insertion of elements (transposable elements). The insertion site of each transposable element was determined by restriction analysis of the derived plasmid (see FIG. 1).

Electroporation: glycerol / sucrose (1
10 ¹⁰ H. pylori cells on blood gelose (10% horse blood) washed in solution (5% v / v and 9% v / v) were taken and resuspended in a volume of 50 μl at 4 ° C. Turned cloudy. Cs
500 ng of plasmid DNA purified on Cl and dialyzed immediately against distilled water was added to the cells in a volume of 1 μl at 4 ° C. After 1 minute on ice, the DNA cells were pre-
Electroporation cuvette cooled to 20 ° C (BioRad r
ef: 165-2086, width 0.2cm), then 25
Placed in the apparatus "Gene Pulser Apparatus-Bio Rad" set to parameters F, 2.5 kv and 200 ohms. After sending out an electrical pulse with a time constant of 4.5-5 msec, 100 μl of SOC buffer (2% bactotryptone, 0.5% bacto-yeast extract, 10 mM Na
Cl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM
The bacteria were resuspended in MgSO ₄ , 20 mM glucose and non-selective blood gelose (without kanamycin but vancomycin, trimethoprim, polymyxin) at 37 ° C. for 48 hours under a microaerobic atmosphere. Nalidixic acid, including amphotericin B). The bacteria were then harvested and 1 volume of Brucella medium (0.5
re-suspended in a selective blood gelose box (2
100 μl of the suspension was spread on the same (containing 0 μg / ml kanamycin and the antibiotic cocktail described above). Growth of transformed, kanamycin-resistant bacteria appears after incubation for 4 days at 37 ° C. in a microaerobic atmosphere. Other techniques, including PCR, the Southern method and the Western method, are conventional.

Results The gene ureB present on the plasmid pILL753
The two mutations generated by the insertion of Mini Tn3-Km into the gene were studied in detail. That is, the mutations numbered 3 and 4. The exact location of each insertion is shown in FIG. Plasmids corresponding to these inserts were prepared, purified and concentrated. H. pylor used for electroporation
bacterial kanamycin resistance showing all the characteristics of the i N6 strain was obtained. These bacteria have no ability to hydrolyze urea.

The control confirmed that the mutant strain was an isogenic strain: ☆ The strain was "urease negative" but H. pyl
It has biochemical properties characteristic of bacteria belonging to ori species (sensitivity to oxidase, catalase and oxygen). ☆ Mother bacteria (N6) (CNCM No. I-1150) and isogenic bacteria N6 :: TnKm-3 and N6 :: Tn
Km-4 has the same restriction profile after enzymatic digestion of total DNA (see FIG. 8). ☆ After enzymatic amplification using H. pylori- specific primers and sequencing of the amplification product, independent strains of H. pylori never show the same sequence, and conversely show significant polymorphisms. , The same nucleotide sequence was found. ☆ Bam HI of DNA of parent strain which has been mutated and
Analysis of the restriction profile by Hind III by Southern-type hybridization demonstrates gene replacement (FIG. 7 and its interpretation FIG. 6).

One of the problems encountered is that the transformed strain (N6) is not the strain from which the urease gene was cloned, this strain is an 85P strain, and the restriction site Hind III and Bam H
I is not conserved from one strain to another: the probe corresponding to the 8.1 kb fragment coming from pILL590 (FIG. 1) clearly shows N6 and 85P
Between different Hind III restriction profiles, in particular 1.
The absence of the 25 kb and 1.15 kb fragments is shown (FIG. 9). In contrast, the 4.1 kb Hind III and 5.1 and 1.3 kb BamHI fragments are conserved. Therefore, the genes ureA , ureB , ure
By enzymatic amplification (PCR) using oligonucleotides distributed over the regions corresponding to C and ureD , the amplification products 1 to 6 shown in FIG. 10 were identical in the two strains, and Hind III restriction The absence of the site was confirmed to reflect a genetic polymorphism rather than a major rearrangement of the urease region. With such confirmation, it is possible to clearly confirm the gene replacement of the wild-type allele by the mutated allele in the two mutants created.

[0145] ☆ Finally, anti H. pylori present in the sera of patients infected by anti H. pylori antibody or H. pylori prepared in the body of the anti-urease antibody or rabbit
By immunoblotting using an antibody, the mutated strains N6 :: TnKm-3 and N6 :: TnKm-4 convert the 61 kD (kilodalton) polypeptide encoded by the gene ureB. It was no longer expressed, thus confirming that the gene ureB of these strains was just disrupted (FIG. 11).

[Brief description of the drawings]

Other advantages and features of the present invention will become apparent from the following examples and figures.

FIG. 1: Transposon subcloning and mutagenesis of pILL753. A : Linear restriction map of the hybrid cosmid pILL585 and plasmid pILL590 (Labigne et al. -199).
1). Gray framework represents the DNA fragment required for expression of urease in C. jejuni. B : Random insertion of transposon Mini Tn3-Km. The numbers (1-24) also correspond to the transposon insertion site in pILL753, similar to the circle; the (+) sign indicates that the transposon did not inactivate urease expression, while the (-) sign indicates This shows that the expression of urease has disappeared. C : hybrid plasmids pILL768 and pIL generated by deletion (△) inside pILL753
L768 linear restriction map. Genes ( ureA to ure)
The localization of H ) is represented by a rectangle. The length of the rectangle is the D.D. required for expressing the polypeptide.
Corresponds to the length of NA. Arrows indicate the direction of transcription. The number of frameworks at the bottom of the figure represents the size of the restriction fragment in kilobases. Numbers in parentheses indicate cloning vectors (pILL575, pILL550 or pI
LL570) H. pylori inserted into one of
Corresponds to the size of the DNA fragment. B, Bam
HI; E, Eco RI, P, Pst I, H, Hind II
I; C, Cla I; Sm, Sma I. The characters in parentheses indicate that the restriction site belongs to the vector.

FIG. 2: E. coli carrying pILL753 as a function of time.
urease activity expressed by Escherichia coli HB101 10 L each in boxes prepared with L agar medium (ML) or M9 minimal medium supplemented with 10 mM L-arginine (MM)
0 μl aliquots of the culture were inoculated and suspended in 0.85% sterile NaCl (10 ⁸ bacteria / ml). Boxes were incubated in (A) 30 ° C. or (B) 37 ° C. in aerobic or microaerobic medium and activity measurements were performed as needed. The asterisk indicates that no urease activity was detected.

FIG. 3 DNA of an auxiliary gene for urease of H. pylori
Sequence A : strategy for sequencing the auxiliary gene of the urease region of the hybrid plasmid pILL753. The arrow is
It corresponds to the size of the sequenced DNA fragment. The head of the arrow performs the oligonucleotide determination,
Indicates an oligonucleotide used for confirmation. B : Schematic representation of the five open reading frames (open reading frames; ORFs) deduced from analysis of the nucleotide sequence and their nucleotide size. ATG corresponds to the start codon for each gene. C : Calculated size and molecular weight of five additional polypeptides of H. pylori urease.

FIG. 4A. Nucleotide sequence of the H. pylori urease accessory gene. The numbers above the sequence represent nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4B. Nucleotide sequence of the H. pylori urease accessory gene. Numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4C. Nucleotide sequence of the H. pylori urease accessory gene. The numbers above the sequence represent nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4D. Nucleotide sequence of the accessory gene for H. pylori urease. The numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4E. Nucleotide sequence of an accessory gene for H. pylori urease. The numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4F. Nucleotide sequence of H. pylori urease accessory gene. Numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4G. Nucleotide sequence of H. pylori urease accessory gene. Numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 4H. Nucleotide sequence of H. pylori urease accessory gene. Numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 41. Nucleotide sequence of H. pylori urease accessory gene. Numbers above the sequence indicate nucleotide positions. In sequence order, the predicted amino acid sequences are: Ur
eI (bp 211 to 795), UreE (bp 800 to 13)
09), UreF (bp 1324-2091), UreG
(Bp 2123-2719) and UreH (bp 2722-bp).
3516). Potential ribosome binding sequence (Shine-Da
lgarno, SD site) is underlined. The boxed sequence corresponds to the promoter-like type (σ54) sequence, and the arrow above the sequence represents a loop-like structure with elements of the rho-independent transcription termination signal [Rosenber
g etal. (1979) Annu. Rev. Genet. 13 : 319-359]. The dotted line below the amino acid sequence corresponds to the binding domain of DNA ( ureI ) or ATP ( ureG ) of the protein [Higgins et al. (1985) EMBO J. 4 : 1033-1040 and Pab
o et al. (1984) Ann. Rev. Biochem. 53 : 293-321].

FIG. 5 is a genetic organization P. mirabilis [Jones et al of the urease operon. (1989) J. Bacteriol. 17
1 : 6414-6422), K. aerogenes (Mulrooney et al.- 1
990) and the relative position of the gene encoding the polypeptide associated with the urease operon of H. pylori . Percentages relate to the percentage of identical amino acids between two similar genes. Blank boxes represent genes unique to the operon.

FIG. 6. Analysis of parental and mutated strains.

FIG. 7: Analysis of parental strains and mutant strains.

FIG. 8. Strain 85P, N6 and mutated N6
(Urease ^-) restriction profile after enzymatic digestion of the total DNA of

FIG. 9. Four ures in the genome of strains 85P and N6
Genomic organization of genes. The DNA-specific fragment is shown in FIG.
By using 8 pairs of primers according to H.p.
chromosome DN extracted from ylori isolates 85P and N6
A was amplified from A. Amplification products were separated by electrophoresis on a 1.4% agarose gel. The value on each side of the gel corresponds to the size (in kilobases) on a 1 kb scale used as a standard.

Four ur in Figure 10 within the genome of the strain 85P and N6
Genomic organization of the e gene. DNA specific fragments by the use of eight pairs of primers according to FIG. 10, H.
chromosome D extracted from pylori isolates 85P and N6
Amplified from NA. Amplification products were separated by electrophoresis on a 1.4% agarose gel. The value on each side of the gel corresponds to the size (in kilobases) on a 1 kb scale used as a standard.

FIG. 11: Immunoblotting using an antibody

FIG. 12. Mutagenesis by transposon: H. pyl
Schematic representation of the four sequential steps required for the construction of a mutant in ori bacteria. Junction 1: Inc containing Transposon Mini Tn3-Km
Group F transferable plasmid pOX38 was transformed into a plasmid pTCA that constitutively expresses transposase Tn3 (TnpA) and is immune to Tn3 in light of the presence of the sequence Tn3-38 bp; An E. coli HB101 comprising a conjugate suicide vector containing a cloned fragment of H. pylori to be induced.
Introduce inside. Kanamycin HB101 transzygous individuals were cultured at 30 ° C. for 48 hours, and the bacteria were E. coli DH1
(Na1). Conjugation 2: Co-integrates resulting from transposition of Mini Tn3-Km in plasmids derived from pILL570 in the absence of resol base were converted to D
Select as conjugated kanamycin co-integrants in H1 cells. Conjugation 3: co-integrate was transposon (pOX38-
Mini Tn3-Km) consisting of primitive donors and Mini T
In the NS2114 strain (Rif) harboring the cre gene, which consists of a hybrid plasmid derived from pILL570 into which n3-Km has been inserted, which allows the specific recombination of the co-integrant into two replicons. Introduce. Positive selection of the isolated form of the co-integrant was achieved with 300 μg / ml kanamycin and 300 μg / ml.
Obtained by selection of transconjugate NS2114 with kanamycin on medium containing g / ml spectinomycin. The final step, consisting of the introduction of H. pylori mutated DNA, is the E. coli NS21 obtained in step 3
14 (reference strain) using the extracted plasmid H. pyl
This can be done by electroporating the ori .

FIG. 13: Seifert et al. (1986, PNAS, USA, 83 : 735-73).
Restriction map of Mini Tn3 according to 9) The asterisk indicates a restriction site in plasmid pILL570 that was changed during vector construction.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/04 A61P 31/04 31/04 35/00 35/00 C12R 1:01 (C12N 9/80 C12N 15/00 ZNAA C12R 1:01) (C12Q 1/68 C12R 1:01) (71) Applicant 502054738 Institute National de la Sante et de la Roussieres Medical INSTITUT NATIONAL D E LA SANTE ET DE LA RECHERCHE MEDICAL (INSERM) France, 75654 Paris Cedu 13, Rue de Torbillac, 101 (72) Inventor Labine, Agnes France, 91,440 Bourg-sur-Yvette, Avenue Beau-Sejour, 47 (72) Inventor Cussack, Valerie France, 75020 Paris, Rue d'Aubron, 59 (72) Inventor Ferrero, Richard France, 75015 Paris, Rue de Beaugiard, 154 F-term ( (Reference) 4B024 AA01 AA11 BA11 CA04 CA09 CA20 DA05 DA06 EA04 EA06 GA11 HA01 HA12 4B050 CC04 DD02 LL10 4B063 QA01 QA19 QQ03 QQ06 QQ44 QR32 QR55 QR62 QS25 QS34 4B065 AA01X AA01Y AA26A01CA03 BA01A14C

Claims (16)

[Claims] 1. One of the nucleic acid sequences shown in FIGS. 4A to 4I.
UreE, ureF, ure
Nucleotides 209-282 of the nucleotide sequence shown in FIGS. 4A-4I in any fragment corresponding to the nucleic acid sequence corresponding to the genes called G, ureH, ureI or a modified sequence thereof, or in any fragment of a portion of the ureI gene Is any part of at least one of these nucleic acid sequences except for the part of the nucleic acid sequence of the ureI gene corresponding to the UreE, UreF, Ure polypeptides such as those expressed by H. pylori.
For the properties of G, UreH or UreI, the functional properties of the polypeptide encoded by the moiety are conserved, attenuated or removed) 2. A polypeptide UreE, UreF, UreG, UreH or Ure which expresses H. pylori.
For the properties of I, whether the functional properties are preserved,
In a form such as being attenuated or absent, or in a form such as a modified sequence that does not express the polypeptide in H. pylori, the functional properties of the polypeptide encoded by the modified sequence are: Has been modified by deletion, addition, substitution or inversion of one or more nucleotides,
An isolated nucleic acid according to claim 1. 3. The mutant is a polypeptide UreE, UreF, UreG, UreH which expresses H. pylori.
Or encodes a polypeptide having functional homology to UreI, or conversely, attenuates and thus eliminates the functional properties of the polypeptide UreE, UreF, UreG, UreH or UreI such that H. pylori is expressed. Or ureE, ure shown in FIGS. 4A-4I, independently modified to encode a modified polypeptide that is no longer expressed as a polypeptide.
3. The nucleotide sequence according to claim 1 or 2, comprising or comprising a set formed by nucleic acid sequences (variants) corresponding to the genes called F, ureG, ureH, ureI. 4. The fragment comprises at least 15 nucleotides, in particular, the fragment comprises: ureE, ureF, ureG in H. pylori
Or a fragment capable of encoding a polypeptide having functional homology to a polypeptide as obtained by expression of a gene selected from ureI, such as expressed from the gene ureE, ureF, ureG, ureH or ureI. A fragment which does not have the ability to encode a H. pylori polypeptide;-ureE, ureF, ureG, ur of H. pylori
3. A fragment of the nucleotide sequence according to claim 1 or 2, wherein the fragment is selected from fragments encoding polypeptides or peptides having properties that are attenuated or even deleted for the properties of the polypeptide encoded by eH or ureI. 5. The method according to claim 1, which is related to a nucleic acid sequence corresponding to the structural genes ureA and ureB encoding the urease subunit in H. pylori.
Item. 6. Genes ureA, ureB, ureC and ureD encoding urease in H. pylori
The nucleotide sequence according to any one of claims 1 to 5, which is related to: 7. A nucleotide probe consisting of the nucleotide sequence according to any one of claims 1 to 6, wherein the sequence is labeled. 8. For use in a gene amplification reaction,
A nucleotide primer comprising a nucleotide fragment as derived from a sequence according to any one of claims 1 to 6, comprising about 18 to about 30, preferably about 25 to about 30 nucleotides. 9. The method of claim 1 under stringent conditions.
Hybridizing with the sequence of any one of claims 1 to 6,
Nucleotide sequence. 10. The polypeptide UreE, UreF, UreG, UreH or Ure shown in FIGS. 4A to 4I.
I corresponds to one of or at least one optional part thereof, and said optional part is a polypeptide UreE, UreF, UreG, U in H. pylori
A polypeptide wherein the properties of the polypeptide in the regulation and / or maturation of urease such that reH or UreI are expressed are attenuated or deleted. 11. The polypeptide Ur in H. pylori
Modified by the addition, substitution, deletion or inversion of one or more amino acids to attenuate and / or delete properties in the control and / or maturation of urease such that eE, UreF, UreG, UreH or UreI are expressed The polypeptide of claim 10, in form. A recombinant vector comprising the sequence according to any one of claims 1 to 6. 13. The recombinant vector according to claim 12, which is a cosmid or a plasmid. 14. The number I-11 on October 3, 1991.
48. The plasmid plLL753 contained in E. coli HB101 deposited at the CNCM at 48.
14. The recombinant vector according to 2 or 13. 15. Number I-11 on October 3, 1991
The plasmid plLL763 contained in E. coli HB101 deposited with the CNCM at No. 49.
14. The recombinant vector according to 2 or 13. 16. The gene ure shown in FIGS. 4A to 4I under conditions which express a urease-negative phenotype or show an attenuated effect of urease, in particular its pathogenic effect.
E, ureF, ureG, ureH and ureI or u
A recombinant showing a mutation at at least one level of a gene selected from reA or ureB
H. pylori strain.